含红芪、黄芪的益气养血汤与补中益气汤含药血清对SAMP8小鼠脾淋巴细胞免疫功能影响的比较研究
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摘要
目的比较分别含红芪、黄芪的益气养血汤和补中益气汤含药血清对SAMP8小鼠脾淋巴细胞免疫功能影响差别。方法 50只青龄昆明种小鼠随机分为益气养血汤红芪组、益气养血汤黄芪组、补中益气汤红芪组、补中益气汤黄芪组、空白组,每组10只,连续灌胃14天,制备含药血清和空白血清。提取SAMP8小鼠脾淋巴细胞,分为益气养血汤红芪含药血清组、益气养血汤黄芪含药血清组、补中益气汤红芪含药血清组、补中益气汤黄芪含药血清组和空白血清组,以相应含药血清和空白血清培养;SAMP8鼠组和青龄鼠组以完全培养液分别培养SAMP8小鼠和青龄小鼠脾淋巴细胞。不同质量分数(20%、40%、80%)含药血清和空白血清培养SAMP8小鼠脾淋巴细24、48、72 h,MTT法检测各组脾淋巴细胞增殖功能,确定最佳含药血清质量分数和作用时间。另取SAMP8和青龄小鼠脾淋巴细胞,4个含药血清组和空白血清组以最佳质量分数血清培养SAMP8小鼠脾淋巴细胞至最佳作用时间,SAMP8鼠组和青龄鼠组以完全培养液分别培养SAMP8小鼠和青龄小鼠脾淋巴细胞48 h,流式细胞仪检测脾T淋巴细胞表面CD28~+、CD152~+分子的表达,ELISA法检测培养上清液中TNF-α的表达,实时荧光定量PCR和Western Blot检测脾T淋巴细胞PI3K mRNA和蛋白的表达。结果 MTT结果显示,当分别含红芪和黄芪的益气养血汤含药血清质量分数为40%培养48 h,分别含红芪和黄芪的补中益气汤含药血清质量分数为40%培养72 h时,SAMP8小鼠脾淋巴细胞增殖效果最佳。与青龄鼠组比较,SAMP8鼠组小鼠脾T淋巴细胞表面CD28~+分子表达下降,CD152~+分子表达增高(P<0.05),TNF-α表达及PI3K mRNA和蛋白表达降低(P<0.05)。与空白血清组比较,分别含红芪和黄芪的益气养血汤和补中益气汤含药血清组小鼠脾T淋巴细胞表面CD28~+分子表达升高,CD152~+分子表达降低(P<0.05),TNF-α表达及PI3K mRNA和蛋白表达均升高(P<0.05)。益气养血汤红芪含药血清组和益气养血汤黄芪含药血清组CD28~+分子表达分别高于补中益气汤红芪含药血清组和补中益气汤黄芪含药血清组(P<0.05)。与益气养血汤黄芪含药血清组比较,益气养血汤红芪含药血清组及补中益气黄芪含药血清组PI3K mRNA表达增高(P<0.05);与补中益气汤红芪含药血清组比较,益气养血汤红芪含药血清组及补中益气汤黄芪含药血清组PI3K mRNA表达降低(P<0.05)。结论含红芪的益气养血汤和补中益气汤含药血清在上调SAMP8小鼠脾T淋巴细胞PI3K mRNA表达上更具优势;含红芪和黄芪的益气养血汤含药血清在上调CD28~+分子表达上优于含红芪和黄芪的补中益气汤含药血清,但在调节PI3K mRNA表达上,含红芪和黄芪的补中益气汤含药血清更具优势。
Objective To compare the different effects between Yiqi Yangxue and Buzhong Yiqi Decoction Containing Serums with Radix Hedysari or Radix Astragali on immunologic function of SAMP8 mice splenic lymphocytes. Methods Fifty young Kunming mice were randomly divided into 5 groups:Yiqi Yangxue Decoction with Radix Hedysari(YYDRH) group, Yiqi Yangxue Decoction with Radix Astragali(YYDRA) group, Buzhong Yiqi Decoction with Radix Hedysari(BYDRH) group, Buzhong Yiqi Decoction with Radix Astragali(BYDRA) group and blank group, 10 in each group. Normal saline and medicine were continuous intragastric administrated for 14 days to prepare drug-containing serums and blank serum. Splenic lymphocytes were divided into 5 groups: YYDRH containing serum group, YYDRA containing serum group, BYDRH containing serum group, BYDRA containing serum group and blank serum group, and were cultured with serums accordingly. The splenic lymphocytes of SAMP8 mice group and young mice group were cultivated with complete medium. To determine the optimal serum concentration and culture time on promoting proliferation of SAMP8 mice splenic lymphocytes, MTT assay was performed after culturing 24 h, 48 h and 72 h with 20%, 40%, 80% concentration of medicated serums and blank serum respectively. SAMP8 mice splenic lymphocytes were exposed with the best concentration of medicated serums and blank serum to optimal culture time. SAMP8 mice group and young mice group were cultured with complete medium for 48 h. Expressions of CD28~+ and CD152~+ molecules on the surface of splenic T lymphocytes were measured by flow cytometry. The level of TNF-α in lymphocytes culture supernatant was tested by ELISA. Real time fluorescence quantitative PCR and Western blot were used to detect expression of PI3K mRNA and its protein. Results MTT results showed that YYDRH/YYDRA and BYDRH/BYDRA containing serums could enhance the proliferation of SAMP8 mice splenic lymphocytes, and the effect was time and dosage dependent. The best efficacy in enhancing lymphocytes proliferation were obtained by cultivating with 40% YYDRH/YYDRA containing serums for 48 h or with 40% BYDRH/BYDRA containing serums for 72 h. Compared with young mice group, CD152~+ level of SAMP8 mice increased, and expressions of CD28~+, TNF-α,PI3K mRNA and its protein decreased(P<0.05). Compared with blank serum group,CD152~+ level of containing serums groups down-regulated, while expressions of CD28~+, TNF-α,PI3K mRNA and its protein up-regulated(P<0.05). The expression of CD28~+ in YYDRH/YYDRA containing serum group was higher than that in BYDRH/BYDRA containing serum group respectively(P<0.05). Compared with YYDRA drug serum group, more expression of PI3K mRNA in YYDRH and BYDRA containing serum group was detected(P<0.05). Besides, it showed fewer expression in YYDRH and BYDRA containing serum group than in BYDRH containing serum group(P<0.05). Conclusions YYD and BYD with Radix Hedysari containing serums are more advantageous in up-regulating the expression of PI3K mRNA in the splenic T lymphocytes of SAMP8 mice. YYDRH and YYDRA containing serums are superior to BYDRH and BYDRA containing serums in increasing the expression of CD28~+ molecule, while this advantage is reversed in regulation the PI3K mRNA.
Objective To compare the different effects between Yiqi Yangxue and Buzhong Yiqi Decoction Containing Serums with Radix Hedysari or Radix Astragali on immunologic function of SAMP8 mice splenic lymphocytes. Methods Fifty young Kunming mice were randomly divided into 5 groups:Yiqi Yangxue Decoction with Radix Hedysari(YYDRH) group, Yiqi Yangxue Decoction with Radix Astragali(YYDRA) group, Buzhong Yiqi Decoction with Radix Hedysari(BYDRH) group, Buzhong Yiqi Decoction with Radix Astragali(BYDRA) group and blank group, 10 in each group. Normal saline and medicine were continuous intragastric administrated for 14 days to prepare drug-containing serums and blank serum. Splenic lymphocytes were divided into 5 groups: YYDRH containing serum group, YYDRA containing serum group, BYDRH containing serum group, BYDRA containing serum group and blank serum group, and were cultured with serums accordingly. The splenic lymphocytes of SAMP8 mice group and young mice group were cultivated with complete medium. To determine the optimal serum concentration and culture time on promoting proliferation of SAMP8 mice splenic lymphocytes, MTT assay was performed after culturing 24 h, 48 h and 72 h with 20%, 40%, 80% concentration of medicated serums and blank serum respectively. SAMP8 mice splenic lymphocytes were exposed with the best concentration of medicated serums and blank serum to optimal culture time. SAMP8 mice group and young mice group were cultured with complete medium for 48 h. Expressions of CD28~+ and CD152~+ molecules on the surface of splenic T lymphocytes were measured by flow cytometry. The level of TNF-α in lymphocytes culture supernatant was tested by ELISA. Real time fluorescence quantitative PCR and Western blot were used to detect expression of PI3K mRNA and its protein. Results MTT results showed that YYDRH/YYDRA and BYDRH/BYDRA containing serums could enhance the proliferation of SAMP8 mice splenic lymphocytes, and the effect was time and dosage dependent. The best efficacy in enhancing lymphocytes proliferation were obtained by cultivating with 40% YYDRH/YYDRA containing serums for 48 h or with 40% BYDRH/BYDRA containing serums for 72 h. Compared with young mice group, CD152~+ level of SAMP8 mice increased, and expressions of CD28~+, TNF-α,PI3K mRNA and its protein decreased(P<0.05). Compared with blank serum group,CD152~+ level of containing serums groups down-regulated, while expressions of CD28~+, TNF-α,PI3K mRNA and its protein up-regulated(P<0.05). The expression of CD28~+ in YYDRH/YYDRA containing serum group was higher than that in BYDRH/BYDRA containing serum group respectively(P<0.05). Compared with YYDRA drug serum group, more expression of PI3K mRNA in YYDRH and BYDRA containing serum group was detected(P<0.05). Besides, it showed fewer expression in YYDRH and BYDRA containing serum group than in BYDRH containing serum group(P<0.05). Conclusions YYD and BYD with Radix Hedysari containing serums are more advantageous in up-regulating the expression of PI3K mRNA in the splenic T lymphocytes of SAMP8 mice. YYDRH and YYDRA containing serums are superior to BYDRH and BYDRA containing serums in increasing the expression of CD28~+ molecule, while this advantage is reversed in regulation the PI3K mRNA.
引文
[1] 国家药典委员会. 中华人民共和国药典[M]. 北京: 中国医药科技出版社, 2015: 993-995.
[2] 马芹颖, 王彦永, 马晓伟, 等. 快速老化小鼠SAMP8的增龄性老化特征研究[J]. 新医学, 2013, 44(6): 415-419.
[3] 邢文婷, 张李峰, 卫东锋, 等. 红芪替代补中益气汤中的黄芪对老龄小鼠脾T 淋巴细胞的影响[J]. 中成药, 2014, 36(3): 450-456.
[4] 张高林, 程卫东, 张文君, 等. 比较含红芪和含黄芪的补中益气汤含药血清对SAMP8鼠脾淋巴细胞抗免疫老化的作用[J]. 中国中药杂志, 2016, 41(15): 2888-2894.
[5] 桂曼曼, 张李峰, 李雪嫣, 等. 比较含黄芪和含红芪的补中益气汤对小鼠免疫功能的影响[J]. 中医杂志, 2012, 53(2): 145-147.
[6] 李纬, 程卫东, 张文君, 等. 含红芪和含黄芪的益气养血汤含药血清对SAMP8小鼠脾脏T淋巴细胞的影响对比分析[J]. 中医杂志, 2016, 57(14): 1237-1241.
[7] 李纬, 程卫东, 张文君, 等. 含红芪、黄芪的益气养血汤对SAMP8小鼠免疫衰老及p38MAPK表达的影响[J]. 中国实验方剂学杂志, 2016, 22(24): 105-110.
[8] 郭菊梅, 杨光, 陈慧昱, 等. 红芪替换黄芪后益气养血汤对老龄鼠抗氧化损伤及抗免疫衰老作用的比较研究[J]. 西部中医药, 2014, 27(8): 7-10.
[9] 杨光, 程卫东, 张李峰, 等. 以红芪替换黄芪的益气养血复方含药血清对老龄小鼠脾淋巴细胞增殖和抗氧化的影响比较[J]. 中药材, 2014, 37(8): 1410-1414.
[10] 徐叔云主编. 药理实验方法学[M]. 北京: 人民卫生出版社, 1982: 178-179.
[11] 田满荣, 杨凤爱. 从中医角度浅释衰老的病因[J]. 陕西中医, 2007, 28(10): 1447-1448.
[12] 张李峰, 蒲建中, 鲍英存, 等. 比较同一复方中用红芪与用黄芪对免疫抑制小鼠淋巴细胞和Th1/Th2型细胞因子的影响[J]. 免疫学杂志, 2012, 28(3): 212-216.
[13] 胡燕, 程卫东, 刘欣, 等. 红芪和黄芪水提物对小鼠免疫功能影响的差异[J]. 北京中医药大学学报, 2011, 34(9) : 623-626.
[14] 李彦红, 刘颖, 秦川. 免疫衰老与T、B细胞改变的相关研究[J]. 中国比较医学杂志, 2012, 22(6) : 65-71.
[15] 许化溪. 前言——免疫衰老与老年疾病[J]. 实用老年医学, 2010, 24(3): 179-180.
[16] 张文君, 程卫东, 李纬, 等. 红芪替换玉屏风散中黄芪对SAMP8小鼠抗免疫老化作用的比较[J]. 中成药, 2016, 38(4): 920-924.
[17] 张高林, 程卫东, 张文君, 等. 比较含红芪和含黄芪的补中益气汤含药血清对SAMP8鼠脾淋巴细胞抗免疫老化的作用[J]. 中国中药杂志, 2016, 41(15): 2888-2894.
[18] 蔡瑞琨. 高免疫原性T细胞抗原表位预测方法的研究[D]. 武汉: 华中科技大学, 2012.
[19] 王文珊, 傅冷西, 叶君健. TNF-α信号传导通路的研究进展[J]. 福建医科大学学报, 2005, 39(S1): 27-31.
[20] 聂颖, 李昌崇. PI3K信号通路对T细胞发育、活化、增殖、分化及支气管哮喘的作用[J]. 中华哮喘杂志(电子版), 2011, 5(5): 348-352.
[21] 蒋龙翔, 俞康. CD28/PI3K在T细胞增殖、活化信号传递中的作用机制[J]. 现代实用医学, 2006, 18(3): 206-209.
[22] 朱光旭, 朱利民. PI-3K/Akt信号传递途径与T细胞反应[J]. 国外医学(免疫学分册), 2004, 27(2): 97-100.
[23] 尹承龙, 劳学军. PI3K-AKT-mTOR信号通路的研究进展[J]. 中国医学创新, 2016, 13(1): 145-148.
[2] 马芹颖, 王彦永, 马晓伟, 等. 快速老化小鼠SAMP8的增龄性老化特征研究[J]. 新医学, 2013, 44(6): 415-419.
[3] 邢文婷, 张李峰, 卫东锋, 等. 红芪替代补中益气汤中的黄芪对老龄小鼠脾T 淋巴细胞的影响[J]. 中成药, 2014, 36(3): 450-456.
[4] 张高林, 程卫东, 张文君, 等. 比较含红芪和含黄芪的补中益气汤含药血清对SAMP8鼠脾淋巴细胞抗免疫老化的作用[J]. 中国中药杂志, 2016, 41(15): 2888-2894.
[5] 桂曼曼, 张李峰, 李雪嫣, 等. 比较含黄芪和含红芪的补中益气汤对小鼠免疫功能的影响[J]. 中医杂志, 2012, 53(2): 145-147.
[6] 李纬, 程卫东, 张文君, 等. 含红芪和含黄芪的益气养血汤含药血清对SAMP8小鼠脾脏T淋巴细胞的影响对比分析[J]. 中医杂志, 2016, 57(14): 1237-1241.
[7] 李纬, 程卫东, 张文君, 等. 含红芪、黄芪的益气养血汤对SAMP8小鼠免疫衰老及p38MAPK表达的影响[J]. 中国实验方剂学杂志, 2016, 22(24): 105-110.
[8] 郭菊梅, 杨光, 陈慧昱, 等. 红芪替换黄芪后益气养血汤对老龄鼠抗氧化损伤及抗免疫衰老作用的比较研究[J]. 西部中医药, 2014, 27(8): 7-10.
[9] 杨光, 程卫东, 张李峰, 等. 以红芪替换黄芪的益气养血复方含药血清对老龄小鼠脾淋巴细胞增殖和抗氧化的影响比较[J]. 中药材, 2014, 37(8): 1410-1414.
[10] 徐叔云主编. 药理实验方法学[M]. 北京: 人民卫生出版社, 1982: 178-179.
[11] 田满荣, 杨凤爱. 从中医角度浅释衰老的病因[J]. 陕西中医, 2007, 28(10): 1447-1448.
[12] 张李峰, 蒲建中, 鲍英存, 等. 比较同一复方中用红芪与用黄芪对免疫抑制小鼠淋巴细胞和Th1/Th2型细胞因子的影响[J]. 免疫学杂志, 2012, 28(3): 212-216.
[13] 胡燕, 程卫东, 刘欣, 等. 红芪和黄芪水提物对小鼠免疫功能影响的差异[J]. 北京中医药大学学报, 2011, 34(9) : 623-626.
[14] 李彦红, 刘颖, 秦川. 免疫衰老与T、B细胞改变的相关研究[J]. 中国比较医学杂志, 2012, 22(6) : 65-71.
[15] 许化溪. 前言——免疫衰老与老年疾病[J]. 实用老年医学, 2010, 24(3): 179-180.
[16] 张文君, 程卫东, 李纬, 等. 红芪替换玉屏风散中黄芪对SAMP8小鼠抗免疫老化作用的比较[J]. 中成药, 2016, 38(4): 920-924.
[17] 张高林, 程卫东, 张文君, 等. 比较含红芪和含黄芪的补中益气汤含药血清对SAMP8鼠脾淋巴细胞抗免疫老化的作用[J]. 中国中药杂志, 2016, 41(15): 2888-2894.
[18] 蔡瑞琨. 高免疫原性T细胞抗原表位预测方法的研究[D]. 武汉: 华中科技大学, 2012.
[19] 王文珊, 傅冷西, 叶君健. TNF-α信号传导通路的研究进展[J]. 福建医科大学学报, 2005, 39(S1): 27-31.
[20] 聂颖, 李昌崇. PI3K信号通路对T细胞发育、活化、增殖、分化及支气管哮喘的作用[J]. 中华哮喘杂志(电子版), 2011, 5(5): 348-352.
[21] 蒋龙翔, 俞康. CD28/PI3K在T细胞增殖、活化信号传递中的作用机制[J]. 现代实用医学, 2006, 18(3): 206-209.
[22] 朱光旭, 朱利民. PI-3K/Akt信号传递途径与T细胞反应[J]. 国外医学(免疫学分册), 2004, 27(2): 97-100.
[23] 尹承龙, 劳学军. PI3K-AKT-mTOR信号通路的研究进展[J]. 中国医学创新, 2016, 13(1): 145-148.